Alcohol dehydrogenases are well established enzymes that catalyze the interconversion of carbonyl compounds and alcohols. See. e.g., Hummel et al., Eur. J. Biochem., 184:1 (1984); Whitesides et al., Angew. Chem. Int. Ed, Engl., 24:617 (1985); Lemiere, "Enzymes as Catalysts in Organic Synthesis" (Schneider, M.P. ed.), D. Reidel Publishing: 1986, pp 19-34; Jones, J.B. et al., in "Applications of Biochemical Systems in Organic Synthesis" (Jones, J.B., et al. eds.), John Wiley and Sons: New York, 1976, pp 248-376; Jones, J.B., "Mechanisms of Enzymatic reactions: Stereochemistry" (Frey, P.A. ed.) Elsevier Science: 1986, 3-14; Jones, J.B., "Enzymes in Organic Synthesis," Ciba Foundation Symposium III, Pitman: London, 1985, pp. 3-14; Keinan et al., J. Am. Chem. Soc., 108:162 (1986); Keinan et al., J. Am. Chem. Soc., 108:3474 (1986); Drueckhammer et al., Enzyme Microb. Technol., 9:564 (1987); Drueckhammer et al., J. Org. Chem., 53:1607 (1988).
The most extensively used and studied alcohol dehydrogenases have been obtained from horse liver, yeast and the bacterium Thermoanaerobium brockii.
Alcohol dehydrogenase action involves the transfer of a hydride between a substrate (an alcohol or an aldehyde or ketone; i.e., a carbonyl, substrate) and a cofactor, which serves as a hydride acceptor or donor. Typically, the cofactor for alcohol dehydrogenase is nicotinamide adenine dinucleotide (NAD), reduced nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADP), or reduced nicotinamide adenine dinucleotide phosphate (NADPH).
NAD and NADP are major electron (e.sup.-) acceptors in the oxidation of molecules. The reactive part of NAD or NADP is the nicotinamide ring.
In the oxidation of a substrate molecule such as an alcohol, that nicotinamide ring accepts a hydride ion and is reduced. As used herein, the phrase "hydride ion" means H.sup.- (a proton associated with two electrons), deuteride (D.sup.-) (a deuterium ion associated with two electrons) or tritide (T.sup.-) (a tritium ion associated with two electrons). These three isotopic hydride ions can also be referred to as .sup.1 H.sup.-, .sup.2 H.sup.- and .sup.3 H.sup.-.
Any of those hydride ions can be used to reduce NAD or NADP. The reduced forms of NAD and NADP are referred to herein as NADH and NADPH, respectively. By way of example, the structure of NADH is shown below. ##STR1##
The two depicted hydrogens bonded to the nicotinamide ring of NADH are designated H.sub.S and H.sub.R. Those designations are used to indicate the spatial orientation of those hydrogens. The H.sub.S hydrogen has the S configuration and the H.sub.R hydrogen has the R configuration.
Where NADH or NADPH serves as a hydride donor for alcohol dehydrogenase activity, the hydride can be either the H.sub.S or the H.sub.R. Conversely, where NAD or NADP serves as the hydride acceptor for alcohol dehydrogenase activity, the added hydride can be either the H.sub.S or the H.sub.R.
Where the added or donated hydride is H.sub.R, the alcohol dehydrogenase is said to act on the pro-R face of the cofactor. Where the added or donated hydride is H.sub.S, the alcohol dehydrogenase is said to act on the pro-S face of the cofactor.
The carbonyl substrates for alcohol dehydrogenase action exist in two potentially diastereotopic forms, where the side chains attached to the carbonyl carbon are different. Two such arrangements are shown in formulae III and IV, below, where the side chain groups X and Y are of different size (i.e. molecular weight) with X&gt;Y. ##STR2##
If the two groups by standard sequence rules have the order X&gt;Y, that face in which the O&gt;X&gt;and Y groups are placed in a plane and arranged in a clockwise manner (formula III, above) is referred to as the re face, That face in which the three groups are similarly placed in a plane and arranged in a counterclockwise manner (formula IV, above) is referred to as the si face.
Alcohol dehydrogenases that follow Prelog's Rule produce alcohols wherein the carbon atom bearing the formed hydroxyl group has the S configuration. Alcohol dehydrogenases that follow Anti-Prelog's Rule produce alcohols wherein the carbon atom bearing the formed hydroxyl group has the R configuration.
In view of the known cofactor and substrate stereoconfigurations, it can be seen that alcohol dehydrogenases can work in one of four ways. Those four possible mechanisms are illustrated below in Scheme 1 and are designated E.sub.1, E.sub.2, E.sub.3 and E.sub.4. ##STR3##
The E.sub.1 mechanism is characterized by specificity for the pro-R hydrogen of the cofactor and addition of a hydride ion to the Si face of a carbonyl substrate. The E.sub.2 mechanism is characterized by specificity for the pro-S hydrogen of the cofactor and addition of a hydride ion to the Si face of a carbonyl substrate. The E.sub.3 mechanism is characterized by specificity for the pro-R hydrogen of the cofactor and addition of a hydride ion to the Re face of a carbonyl substrate. The E.sub.4 mechanism is characterized by specificity for the pro-S hydrogen of the cofactor and addition of a hydride ion to the Re face of a carbonyl substrate.
The previously described alcohol dehydrogenases from horse liver, yeast and Thermoanaerobium brokii are all characterized as operating via the E.sub.3 mechanism (i.e., they catalyze the transfer of a hydride ion from the pro-R face of the cofactor to the Re face of a carbonyl substrate to produce an alcohol having the S configuration. Prelog, Pure Appl. Chem., 9:119 (1964).
An alcohol dehydrogenase has been isolated from Mucor javanicus and found to operate via the E.sub.2 mechanism.
Recently, an alcohol dehydrogenase enzyme has been isolated from Pseudomonas sp. strain SBD6. That enzyme was found to operate via the E.sub.1 mechanism.
Alcohol dehydrogenases can be further characterized by their specificity for certain cofactors and their ability to act on substrates of varying structural complexity. In this regard, alcohol dehydrogenases can, typically, use either but not both of NAD(H) or NADP(H).
The alcohol dehydrogenases from Pseudomonas sp. strain SBD6, horse liver, yeast and Thermoanaerobium brokii require that the carbonyl or hydroxyl group of the substrate be adjacent to a methyl group. The Lactobacillus kefir alcohol dehydrogenase can use such methyl-substituted substrates, but this enzyme is not limited to such substrates. Rather, this enzyme can use substrates having aromatic, aliphatic and cyclic side chains. Hummel, Appl. Microb. Biotechnol. 34:15 (1990).
In view of the foregoing, it can be seen that there is no predictable relationship between carbonyl substrate structural specificity and hydride transfer mechanism for known alcohol dehydrogenases.